Hepatitis A continues to cause sporadic cases of infection, endemics, occasional deaths and is a public health problem all over the world. The infection is caused by Hepatitis A Virus (HAV) a member of the picornavirus family, a group of small non-enveloped RNA viruses. The virus particle is 27-32 nm in diameter and is composed of three polypeptides cleaved from a single polypeptide precursor molecule. The mature virus is composed of polypeptides VP1, VP2 and VP3. The capsid proteins VP1 and VP3 contain the major antigenic sites and are capable to induce neutralizing antibodies (Lemon et al., 1989, In: Semler et al. eds. Molecular aspects of picornavirus and detection. Washington, D.C.: ASM p 193-208).
Hepatitis A Virus (HAV) is the only hepatotropic virus which can be isolated from cell culture, but the virus is usually difficult to propagate, with long incubation periods and no cytopathic effect. Binn et al. (1984. J. Clincal. Microbiol. 20: 28-33) tested several primate cell types for replication of HAV and optimal conditions for isolation and production of large quantities of virus. Serum free production of HAV was shown in BSC-1 cells, a heterodiploid cell line that until now has not been used for preparation of vaccines for use in humans. After 21 days of culture in roller flasks, virus antigen could be found in the supernatant and the cell fraction. Cells maintained in serum free medium supported viral growth equal to that of cells maintained in serum. A candidate HAV vaccine was obtained from cells and supernatant fluid of infected BSC-1 cells maintained in serum free medium (Binn et al., 1986. J. Infect. Diseases 153: 749-756). However, Simmonds et al. (1985, Appl. Enviromental Microbiol. 49:749-755) found no significant difference of HAV production at different concentration of serum between 2% and 15% in the medium with persistently infected cells BSC-1 or AGMK cells. Virus production in primary AGKM cells was twice that in BSC-1 cells, but HAV produced remained predominantly cell associated and only some virus was found in the culture fluid. Nasser et al. (1987, Appl. Enviromental Microbiol. 53:2967-2971) reported that about seven times more HAV was produced in FRhK-4 cells culture in one-half or less the time that was required for BS-C1 cultures, wherein the ratio of cell-associated HAV versus HAV released BSC-1 cells was calculated to be 80% to 20%, respectively.
Flehmig et al. (1987. J. Medical Virol. 22:7-16) prepared HAV from cell culture supernatant of persistently infected normal human embryonic fibroblasts grown in serum containing medium. Using these methods, large amounts of supernatants were produced in NUNC cell factories and HAV antigen isolated from the supernatant and purified in multiple steps was used for vaccination tests.
Even though several primate cell types have been reported to support replication of HAV, such as fetal rhesus monkey kidney cell line (FRhk-4), primary African green monkey kidney cells (AGKM), continuous African green monkey kidney cells (BCS-1), these cells are generally not used for human vaccine because it is known that monkey kidneys often have high content of latent simian viruses. Other cell lines cannot be used because of the tumorigenic nature of these cells. Mass production of primary human epithelial, fibroblast or kidney cells or cell strains to propagate HAV is also limited by the low passage number of these cells in culture. In fact, the applicable guidelines of the World Health organization (WHO) indicate that only a few cell lines are allowed for virus vaccine production.
One of the cell lines which is currently accepted and validated for the production of vaccine applicable to humans is VERO cells. VERO cells are a continuous monkey kidney cell line that has been licensed for use in the manufacture of human vaccines and is currently used for the production of poliomyelitis and rabies vaccine. Attempts have also been made to use VERO cells for HAV production, but it has been found that replication of HAV on VERO cells is limited because VERO has a temperature restriction of viral growth. In addition, virus is never found in the supernatant fluids of infected cells. (Locarnini et al., 1981, J. Virol. 37: 216-225). U.S. Pat. No. 4,783,407 discloses the production of HAV on VERO cells in roller bottles at a temperature no higher than 33° C. to overcome the temperature restriction. HAV antigen was obtained by freeze-thawing of the cultured cells and release of intracellular produced virus. A commercial vaccine based on propagation of HAV on VERO cells has never been described.
So far, formalin inactivated HAV vaccines have been produced for clinical trials (Andre et al., 1990, In: Melnick (ed): Prog. Med. Virol. Basel, Karger 37: 72-95, Armstrong et al, 1993, J. Hepatology 18: 20-26) and two are commercial available, which induce long-lasting immunity and protection from primary infection. The manufacturing process of the currently available inactivated HAV whole virus vaccines uses the human embryonic lung fibroblast cell line MRC-5 as host cells in Nunc Cell Factories (NCF), wherein the HAV antigen used for vaccine production is obtained form the cell lysate of intracellularly produced virus, because HAV antigen is not efficiently released into the culture supernatant and methods to concentrate the large volume are costly (Bishop et al., 1994. J. Virol. Meth. 47:203-216). HAV large scale preparations from the cell lysates and the cell culture supernatants contain mixed populations of virions and provirions (Bishop et al., 1997. Arch. Virol. 142:2147-2160) and the commercial available vaccine comprises complete mature virions and empty provirion particles (Andre 1990 supra, Armstrong 1993 supra). Moreover, MRC-5 cells grow slowly in tissue culture and require fetal calf serum.
The problems arising from the use of serum in the cell culture and/or protein additives derived from an animal or human source (e.g., the varying quality and composition of different batches and the risk of contamination with mycoplasma, viruses or BSE-agents) are well known. In general, serum or serum derived substances like albumin, transferrin or insulin may contain unwanted agents that can contaminate the cultures and the biological products derived from them. Furthermore, human serum derived additives have to be tested for all known viruses, like hepatitis or HIV, which can be transmitted by serum. Bovine serum and products derived therefrom, for example trypsin, bear the risk of BSE-contamination. In addition, all serum derived products can be contaminated by unknown agents. Therefore, many attempts are being made to provide efficient host systems and cultivation conditions that do not require serum or other serum derived compounds.
The production process is as important as the medium. The only process which is economically feasible is a reactor process because the scale-up can be made appropriate to the market size and the vaccine doses needed. For adherent cells the carrier process with a classical microcarrier is currently the best choice for large scale cultivation of the cells needed for virus propagation. Current processes based on microcarrier culture allow production of viral antigen using fermenter sizes of up to several thousand liters.
Widell et al.(1984, J. Virol. Methods 8:63-71) used microcarrier cell culture systems of FRhk-4 cells for large scale production of HAV and found intra-and extracellular virus. Virus production per cell using the microcarrier system was similar to a conventional culture grown in flask. On the other side, Junker et al (1992, Cytotechnol. 9:173-187) showed that HAV infected MRC-5 cells bound to conventional Cytodex microcarriers only yielded 30% HAV antigen compared to cells grown in flasks because of the tendency of MRC-5 cells to form microcarrier and cell aggregates. WO 95/24468 discloses MRC5 cells grown on aggregated glass-coated microcarriers for HAV production in a perfusion system, wherein the bulk of virus is found in the cells. In the system described, higher concentrations of serum between 2-10% allowed greater production of HAV than at low level concentration of 0.5-2% of serum. However, when Aunins et al. (1997, In: Carrondo et al. (eds), Animal Cell Technology, p.175-183) compared different manufacturing technologies such as Nunc Cell Factories (NCF), microcarriers, static mixed reactors and CellCubes, they found that glass-coated microcarriers as described in WO 95/24468 allowed the formation of stable aggregates and production of HAV. The monodisperse microcarrier suspensions, however, could not be maintained for the duration of the culture, and productivity of the glass aggregate microcarrier process was approximately half of static culture under similar conditions. Aunins et al. 1997 (supra) concluded that a microcarrier culture of the HAV strain used was not feasible.
The worldwide market demand for HAV vaccines is in the order of 100 Million doses per year. Efficient vaccine production requires the growth of large-scale quantities of virus produced in high yields from a host system. The process and cultivation conditions under which a virus strain is grown is of great significance with respect to achieving an acceptable high yield of the strain. Thus, in order to maximize the yield of the desired virus, both the system and the cultivation conditions must be adapted specifically to provide an environment that is advantageous for the production of the desired virus. Therefore, a continuing need exists for safe and effective methods to produce viruses and antigen. Moreover, there is a need for an approach to viral propagation, employing materials that are already available and requiring a minimal number of time-consuming manipulations, wherein the selection of a combination of host cells, culture medium, growth conditions and production system is essential to achieve an efficient production process.